This application relates to lymphokines. In particular, it relates to lymphotoxin and derivatives thereof.
Lymphotoxin was first identified as a biological factor with anticellular activity on neoplastic cell lines. An activity identified as lymphotoxin and obtained from mitogen-stimulated lymphocytes is associated with a spectrum of cytotoxic activities ranging from cytostasis of certain tumor cell lines to marked cytolysis of other transformed cells. However, lymphotoxin activity is characterized by little or no anticellular activity on primary cell cultures and normal cell lines tested. This putative discriminating anticellular property of lymphotoxin led to in vivo studies which suggest that lymphotoxin may have a potent antitumor activity.
Lymphotoxin is the term applied to what has been described as a family of molecules. Lymphotoxin molecules have been identified as glycoproteins divided into five molecular weight classes, each of which in turn is heterogenous with respect to charge. The human alpha (MW 70-90,000) and beta (MW 25-50,000) classes appear to predominate in most lymphocyte supernatants. The alpha MW classes can be separated by charge into at least seven subclasses, while the beta subclass has been separated into two distinct subclasses (G. Granger et al. in Mozes et al., Ed., 1981, Cellular Responses to Molecular Modulators pp 287-310). Also identified have been complex (MW &gt;200,000) and gamma (MW 10-20,000) lymphotoxin forms. The various lymphotoxin forms and classes differ from one another in their stability and kinetics of appearance in culture. Furthermore, they may aggregate together with the complex class under conditions of low ionic strength. The lower molecular weight classes of lymphotoxins have been disclosed to be relatively unstable and weakly cell lytic compared to the higher molecular weight classes (Hiserodt et al., 1976, "Cell. Immun." 26: 211; Granger et al. in De Weck et al. Ed., 1980 Biochemical Characterization of Lymphokines pp 279-283). Gamma class activity has not been studied extensively because of its instability (G. Granger et al., 1978 "Cellular Immunology" 38: 388-402). The beta class also has been reported to be unstable (Walker et al., "J. of Immun." 116[3]: 807-815 [March 1976]).
It should be understood that lymphokine terminology is not uniform. At present, the names given to cell culture products are largely a function of the cells which are believed to elaborate the product and the performance of the products in biological assays. However, these products remain poorly characterized in large measure because many studies have been conducted with partially pure preparations and because the assays used to characterize the products are not molecule-specific and in any case are subject to considerable variation. The true identity of the various cytotoxic factors will remain unknown in the absence of standard terminology based on clearly assayable distinguishing characteristics such as amino acid sequences or immune epitopes. As examples of other names given to cytotoxic cell culture products are tumor necrosis factor, NK cell cytotoxic factor, hemorrhagic necrosis factor and macrophage cytotoxin or cytotoxic factor.
Copending and commonly assigned U.S. Ser. No. 06/608,316, filed May 7, 1984, now abandoned and EP 100,641A (published Feb. 15, 1984) describe amino acid sequences for a human lymphotoxin isolated from the human lymphoblastoid cell line RPMI-1788.
Hayashi et al., EP 132,125A (published Jan. 23, 1985) describe recovering a protein from a rabbit following stimulation of its reticuloendothelial system. The protein was reported to have antitumor activity and the N-terminal amino acid sequence Ser-Ala-Ser-Arg-Ala-Leu-Ser-Asp-Lys-Pro-Leu-Ala-His-Val-Val-Ala-Asn-Pro-Gl n-Val-Glu-Gly-Gln-Seu-Gln-Trp-Leu.
Copending and commonly assigned U.S. Ser. No. 628,059, filed Jul. 5, 1984, now abandoned discloses the purification and recombinant synthesis of a cytotoxic human polypeptide identified as tumor necrosis factor and having the N-terminal amino acid sequence Val-Arg-Ser-Ser-Ser-Arg-Thr-Pro-Ser-Asp-Lys-Pro-Val-Ala-His-Val-Val-Ala-As n-Pro.
Ohnishi et al. (U.S. Pat. No. 4,481,137) discloses obtaining a 7-9,000 MW substance named CB.sub.x3 from BALL-1 cell culture that suppresses the growth of tumor cells and that has an Ala-Ala N-terminus.
According to Toth and Granger, "Mol. Immun." 16: 671-679 (1979), neither the removal of sialic acid from lymphotoxin-containing lymphocyte supernatants by neuraminodase treatment nor the addition of N-acetyl-glucosamine, galactose, lactose, mannose, .alpha.-methyl-mannoside or fucose to the supernatants had any affect on in vitro lytic activity. Toth et al. thus concluded that simple sugars do not appear to play a role in the activity of their lymphotoxin. However, Toth et al. also observe that saccharides play an important role in the action of other lymphokines and concluded that they could not exclude the participation of more complicated forms of oligo saccharides in the cytotoxic activity of lymphotoxin.
Subsequently, Proctor, Klostergaard and Granger ("Clinical Research", 1982, 30(1): 55A) reported that human lymphocytes, when activated by PHA in the presence of tunicamycin (to inhibit the addition of N-linked carbohydrate moieties to lymphotoxin molecules), released biologically inert lymphotoxin. According to these authors, immunochemical studies revealed that while the carbohydrate moiety of lymphotoxin was not needed for its transport and release by the activated lymphocyte into the supernatant, the carbohydrate was needed in order to have effective target cell destruction because the carbohydrate was responsible for the appropriate conformation of the lymphotoxin molecule(s).
Other literature that should be studied in connection with this application includes Evans, "Cancer Immunol. Immunother." 12: 181-190 (1982); Lee et al., "Cell. Immun." 48: 166-181 (1979); De Weck et al. Ed., (1980) Biochemical Characterization of Lymphokines pp 279-312; Khan et al. Ed. (Jun. 30, 1982) Human Lymphokines pp 459-477; Aggarwal et al., Presentation at the 3rd International Lymphokine workshop in Haverford, Pa., Aug. 1-5 1982; Ransom et al., "Cancer Research" 43: 5222-5227 (Nov. 1983); Kull et al., "J. of Immun." 126(4): 1279-1283 (April 1981); J. Sawada, et al., "Jpn. J. Exp. Med." 46: 263-267 (1976); G. Granger et al., "Cell. Immunol." 38: 388-402 (1978); J. Rundell et al., "Immunopharmacology" 3: 9-18 (1981); G. Granger et al., "J. Lymphokine Res." 1: 45-49 (1982); N. Ruddle et al., "Lymphokine Res." 2: 23-31 (1983); M. Mitsuhashi et al., U.K. Patent Application 2,106,117; H. Enomoto, European Patent Application 87,087A; B. Williamson et al., "P.N.A.S. USA" 80:5397-5401 (1983) and S. Wright et al., "J. Immunol." 126: 1516-1521 (1981).
The lymphotoxin (or substances identified as lymphotoxin) obtained heretofore from lymphocyte culture are present in low concentrations, on the order of 0.05-2.times.10.sup.6 units/l in supernatants of RPMI-1788 cells or primary lymphocytes. The amounts harvested often vary considerably, and primary lymphocytes are expensive. An economical method for producing lymphotoxin is needed (Yamamoto et al., "J. of Biological Response Modifiers" 3:[1] 76-87 [1984]).
Prior methods also fail to produce lymphotoxin which is homogeneous as to amino acid sequence, an important feature for drug utilities. Lymphotoxin recovered from cell line culture exhibits amino terminal heterogeneity, probably due to proteolytic processing (see the above cited U.S. Ser. No. 06/608,316). Cultures of primary lymphocytes, e.g. from adenoids or peripheral blood, must necessarily contain the cells of many donors for reasons of economy. However, the products of these cells will reflect genetic variation among the donors so that the resulting "lymphotoxin" may in fact be a mixture of allelic species. Obviously, the proportions and identities of such alleles will be unknown from lot-to-lot. A method is needed for producing lymphotoxin that is uniform as to its amino acid sequence.
Prior methods also are limited to the production of lymphotoxin having primary amino acid sequences corresponding to those found in nature. Substituting, deleting or inserting different amino acids in these sequences would require extensive and costly chemical modifications, if such could be accomplished at all. Methods are needed for easily introducing variations into the amino acid sequences of lymphotoxin.
Although the antitumor effects and apparent therapeutic value of lymphotoxin activity have been reported in the literature since 1968,lymphotoxin has not been studied in extensive clinical protocols or commercialized due to the small quantities and heterogenous nature of the lymphotoxin made available through prior methods. Methods are needed to economically prepare quantities of lymphotoxin adequate for clinical studies.
Rabbit antisera have been described in the literature which are capable of neutralizing the cytolytic activity of various cytotoxins, including substances identified as lymphotoxin (Yamamoto et al. "Cell. Immun." 38: 403-416 (1978); Gately et al., "Cell. Immun." 27: 82-93 (1976); Hiserodt et al., "J. of Immun." 119(2): 374-380 (1977); Zacharchuk et al., "P.N.A.S. USA" 80: 6341-6345 (October 1983); Ruddle et al., "Lymphokine Research" 2(1) 23-31 (1983); Mannel et al., "Infection and Immunity" 33(1): 156-164 (1981); Wallach et al. E. De Maeyer et al. Ed. The Biology of the Interferon System pp 293-302 (Pub. September 1983) and Stone-Wolff et al., "J. Exp. Med." 159: 828-843 (March 1984). Since these antisera are polyclonal it contains a multiplicity of antibodies directed against the immunogen lymphotoxin. Any one or more of these antibodies is acting to neutralize the "lymphotoxin" activity. Further, the literature reports generally are unclear as to the molecular identity of the substance responsible for lymphotoxin activity that was used as the immunogen. What is needed for diagnosis and immunoaffinity purification procedures is a monospecific antibody directed against a clearly and unambiguously identified lymphotoxin molecule. It is an objective of this invention to provide such an antibody.
It is a further object herein to provide methods for economically synthesizing a lymphotoxin form in a composition wherein the primary amino acid sequence of substantially all of the lymphotoxin molecules is the same.
It is another object to produce predetermined variations in the amino acid sequence of a lymphotoxin form, more specifically, amino acid deletions, insertions, substitutions, or combinations thereof.